Metformin enhances insulin signalling in insulin-dependent and-independent pathways in insulin resistant muscle cells

Abstract

1 Metformin lowers blood glucose levels in type 2 diabetic patients. To evaluate the insulin sensitizing action of metformin on skeletal muscle cells, we have used C2C12 skeletal muscle cells differentiated in chronic presence or absence of insulin. 2 Metformin was added during the last 24 h of differentiation of the C2C12 myotubes. Insulin-stimulated tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) was determined. 3 Chronic insulin treatment resulted in 60 and 40% reduction in insulin-stimulated tyrosine phosphorylation of IR and IRS-1, respectively. Treatment with metformin was able to increase the tyrosine phosphorylation of IR and IRS-1 by 100 and 90% respectively. 4 Chronic insulin treatment drastically reduced (45%) insulin-stimulated phosphatidyl inositol 3-kinase (PI 3-kinase) activity. Metformin treatment restored PI 3-kinase activity in insulin-resistant myotubes. 5 Insulin-stimulated glucose uptake was impaired in chronically insulin-treated myotubes. Metformin increased basal glucose uptake to significant levels (P<0.05), but metformin did not increase insulin-stimulated glucose transport. 6 All the three mitogen-activated protein kinases (MAPK) were activated by insulin in sensitive myotubes. The activation of p38 MAPK was impaired in resistant myotubes, while ERK and JNK were unaffected. Treatment with metformin enhanced the basal activation levels of p38 in both sensitive and resistant myotubes, but insulin did not further stimulate p38 activation in metformin treated cells. 7 Treatment of cells with p38 inhibitor, SB203580, blocked insulin- and metformin-stimulated glucose uptake as well as p38 activation. 8 Since the effect of metformin on glucose uptake corresponded to p38 MAPK activation, this suggests the potential role p38 in glucose uptake. 9 These data demonstrate the direct insulin sensitizing action of metformin on skeletal muscle cells.

Figure 1

Effect of insulin stimulation and metformin on tyrosine phosphorylation of IR-β in C2C12 myotubes. Metformin (400 μM) was added during the last day of differentiation for 24 h to C2C12 cells differentiated in absence (MF) or in chronic presence of insulin (MFI). Then, the cells were stimulated with 100 nM of insulin for 5 min and lysed. Cell lysate were immunoprecipitated (IP) with antibodies against IR-β and Western immunoblotted (IB) with anti-phosphotyrosine antibody (A). The blots were stripped and reprobed with IR-β (B). Experiments were repeated thrice and representative blots are shown. Phosphorylation levels of IR (C) were quantified by densitometry and expressed relative to MF (control) samples. Error bars represent s.e.m. of three independent experiments (^*P<0.05).

Figure 2

Effect of insulin stimulation and metformin on tyrosine phosphorylation of IRS-1 in C2C12 myotubes. Samples were treated as described in Figure 1. Cell lysate were immunoprecipitated (IP) with antibodies against IRS-1 and Western immunoblotted (IB) with anti-phosphotyrosine antibody (A). The blots were stripped and reprobed with IRS-1 (B). Experiments were repeated thrice and representative blots are shown. Phosphorylation levels of IRS-1 (C) were quantified by densitometry and expressed relative to MF (control) samples. Error bars represent s.e.m. of three independent experiments (^*P<0.05).

Figure 3

Effect of insulin stimulation and metformin on PI 3-kinase activity in C2C12 myotubes. C2C12 cells, differentiated in MF or MFI, were washed with KRP buffer as described in Methods, followed by stimulation with insulin (100 nM) for 10 min. PI 3-kinase activity in anti-IRS-1 immunoprecipitates was measured as described under Methods. Cells were treated with 400 μM metformin during last 24 h of differentiation. A representative autoradiogram for two independent experiments is shown (A). Relative density against control (MF) of PI3P spots (B) was quantified by densitometry and expressed as an average of two experiments. PI3P refers to PI 3-phosphate.

Figure 4

Effect of insulin stimulation and metformin on 2-DOG uptake in C2C12 myotubes. C2C12 cells, differentiated in MF or MFI, were washed with KRP buffer as described in Methods, followed by stimulation with insulin (100 nM) for 15 min. [³H]-2-DOG uptake was measured as described in Methods. Cells were treated with 400 μM of metformin during last 24 h of differentiation. Error bars represent s.e.m. of four independent experiments (^*P<0.05).

Figure 5

Effect of insulin stimulation and metformin on ERK, JNK and p38 activation: C2C12 cells, differentiated in MF or MFI, were washed with KRP buffer as described in Methods, followed by stimulation with insulin (100 nM) for 5 min. Lysates were prepared from cells treated with 400 μM of metformin during last 24 h of differentiation. Proteins were resolved by SDS–PAGE and immunoblotted either to phospho-specific ERK (A), phospho-specific JNK (D), phospho-specific p38 (G), or ERK (B), JNK (E), p38 (H) antibodies. Phospho-specific blots are representative of experiments performed three times. Error bars of quantified data for pERK (C), pJNK (F) and pp38 (I) represents s.e.m. of three independent experiments (^*P<0.05, ^**P<0.01).

Figure 6

Effect of SB203580 on 2-DOG uptake and p38 activation. C2C12 cells, differentiated in MF or MFI, were incubated in KRP buffer for 30 min followed by another incubation of 30 min in presence of 10 μM of SB203580. Cells were stimulated with 100 nM of insulin in presence of SB203580, followed by (A) immunoblot analysis of p38 activation or (B) 2-DOG uptake for 10 min. Cells were treated with 400 μM of metformin during last 24 h of differentiation. Error bars represent s.e.m. of three independent experiments. A representative blot of pp38 is shown.

Figure 7

Effect of chronic presence of SB203580 on stimulation of 2-DOG uptake and p38 activation by metformin. C2C12 cells, differentiated in MF or MFI, were incubated in KRP buffer twice for 30 min each in presence of 10 μM of SB203580. Cells were stimulated with 100 nM of insulin in presence of SB203580, followed by (A) immunoblot analysis of p38 activation or (B) 2-DOG uptake for 10 min. Cells were treated with 10 μM of SB203580 with or without 400 μM of metformin during last 24 h of differentiation. Error bars represent s.e.m. of three independent experiments. A representative blot of pp38 is shown from three independent experiments.